Difference between revisions of "Part:BBa K2797004:Design"
(→‎Design Notes)
Line 7: Line 7:
  
 
===Design Notes===
 
===Design Notes===
−
An mNeonGreen construct was designed for use as an alternate fluorescent reporter for each test device - replacing GFP. The mNeonGreen sequence was codon optimised using Benchling and Gibson ends were designed using NEBuilder for cloning into pSB1C3. The subsequent sequence was synthesised by IDT. Prior to the PCR linearisation, pSB1C3 plasmid concentration for each mini-prepped test device was determined using a Qubit fluorometer and diluted to 0.5 ng/µl. The diluted pSB1C3 vectors were linearised using a 2 step PCR system following a Q5 Polymerase protocol (NEB). This protocol utilised forward and reverse primers with Tm values of 72°C. The mNeonGreen primers were designed by using the NEB Tm calculator and Benchling. The amplified DNA was then digested with DpnI, heat treated to inactivate the enzyme and assembled via Gibson Assembly using the NEBuilder HiFi DNA Assembly Kit. Following their protocol, a 2-fragment reaction with 0.5 pmol of DNA in a 2:1 insert to vector ratio was done and transformants were plated onto agar plates with the appropriate antibiotic (LB+cam for each test device and LB+amp for the controls).
 
  
 +
[[Image: mneon.png|thumb|right|400px| '''A simulated Gibson Assembly of the mNeonGreen fluorescent protein sequence into the pSB1C3 vector backbone.''' As shown, the mNeonGreen slots into the regions in between the biobrick prefix and suffix, a region where the test device is usually present. The green regions show the mNeon green insert. The mNeongreen gene uses the promoter, RBS and terminator of the test device.]]
 +
 +
Since the idea was to replace the GFPmut3b reporter gene in each of the test device pSB1C3 with the mNeonGreen construct, removal of the GFPmut3b coding region from each vector was required - this was done using 2-step PCR. Six reverse primers complimentary to each of the test device RBS and their varying promoter regions, and 1 a single forward primer to bind at the beginning of the terminator were utilised in 2-step PCR to linearise the respective pSB1C3 vectors - removing GFPmut3b. The resulting amplification was treated with DpnI.
 +
 +
The mNeonGreen sequence was codon optimised for expression in E. coli DH5-alpha using Benchling. Further to this, Gibson ends were designed using NEBuilder for cloning into the linearised pSB1C3 and added to the 3' and 5' ends of the mNeonGreen sequence. The mNeonGreen sequence was synthesised by IDT. The sequence was cloned into each pSB1C3 vector via Gibson Assembly.
  
 
===Source===
 
===Source===

Revision as of 12:55, 10 October 2018


Test Device 1 for the iGEM InterLab Study (mNeonGreen)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 100
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

A simulated Gibson Assembly of the mNeonGreen fluorescent protein sequence into the pSB1C3 vector backbone. As shown, the mNeonGreen slots into the regions in between the biobrick prefix and suffix, a region where the test device is usually present. The green regions show the mNeon green insert. The mNeongreen gene uses the promoter, RBS and terminator of the test device.

Since the idea was to replace the GFPmut3b reporter gene in each of the test device pSB1C3 with the mNeonGreen construct, removal of the GFPmut3b coding region from each vector was required - this was done using 2-step PCR. Six reverse primers complimentary to each of the test device RBS and their varying promoter regions, and 1 a single forward primer to bind at the beginning of the terminator were utilised in 2-step PCR to linearise the respective pSB1C3 vectors - removing GFPmut3b. The resulting amplification was treated with DpnI.

The mNeonGreen sequence was codon optimised for expression in E. coli DH5-alpha using Benchling. Further to this, Gibson ends were designed using NEBuilder for cloning into the linearised pSB1C3 and added to the 3' and 5' ends of the mNeonGreen sequence. The mNeonGreen sequence was synthesised by IDT. The sequence was cloned into each pSB1C3 vector via Gibson Assembly.

Source

Sequence from Allele Biotechnology

References